Cancer chemotherapy utilizing the 2-chloroethylnitrosoureas is drug toxicity dose limiting. The principal clinical toxicity is a delayed depression of bone marrow function. Both the mechanism(s) for toxicity and for anti-tumor activity are poorly understood. The very rapid metabolism of these drugs and the delayed toxicity is somewhat paradoxical. Current theories contend that the spontaneous chemical breakdown of the nitrosoureas generates reactive intermediates that are ultimately responsible for cell death. Because the half-life of these compounds in plasma and the half-life for killing tumor cells are both much lower than the chemical half-life, it raises question regarding the relative roles of chemical breakdown versus metabolism in the expression of toxicity and anti-tumor activity. It is proposed to study the metabolism of selected 2-chloroethylnitrosoureas in vivo and in vitro with the view that a more detailed knowledge of metabolism will lead to a better understanding of the underlying mechanisms, which will hopefully lead to design of new and better drugs for clinical use. Metabolism will be studied using nitrosoureas labeled with stable isotopes (2H, 13C, 15N) in combination with radioisotopic compounds to enhance opportunity of identifying metabolites by liquid chromatography/mass spectrometry and NMR. Efforts to utilize metabolite structural information to deduce the nature of reactive intermediates that could account for the biological activities will be made. It is proposed to identify the alkylating carbon of the 2-chloroethyl moiety by use of 13C-labeled precursors and a combination of mass spectrometry and 13C-NMR. The mechanism of denitrosation will be studied. It is possible that new reactive functional groups such as N-hydroxy, N-amino or N-nitro are formed, in which case their role in nitrosourea action would be opened for study. Liver nuclear metabolism of the drugs will be studied and if it occurs, nuclei will offer a good model for comparison of the relative roles of chemical degradation and metabolism in their biological expression.